Filter Report from CaMutQC
2025-06-25
Overview
There are originally 87 mutations, 46 left after potential artifacts filtration, 12 left after candidate mutation selection. TMB Calculation was not asked.
Settings
Metadata
- Tumor sample name: WES_EA_T_1
- Normal Sample name: WES_EA_N_1
- Number of rows in the final MAF data frame: 87
Report configuration
1 Potential artifacts filtration
a. Sequencing quality thresholds
- tumor total depth (tumorDP): 20
- tumor allele depth (tumorAD): 5
- normal total depth (normalDP): 10
- VAF: 0.05
- VAF ratio (tumorVAF/normalVAF): 0
b. Strand of Bias
Strand bias occurs when the genotype inferred from information presented by the forward strand and the reverse strand disagrees. A study showed that post-analysis procedures can cause strand bias, which introduce more SNPs with higher strand bias, and in turn results in more false-positive SNPs 1. Therefore, it is necessary to detect and minimize the strand bias of our data.
At present, there are four methods of strand bias detection that are widely used. In a mitochondria heteroplasmy study 2, the calculation of SB was put forward. GATK calculates a strand bias score for each SNP identified, and Samtools also computes a strand bias score based on Fisher’s exact test. Additionally, GATK introduced an updated form of the Fisher Strand Test, StrandOddsRatioSOR annotation, which is believed to be better at measuring strand bias for data in high coverage.
In CaMutQC, either Fisher Strand Test or SOR algorithm can be used to evaluate strand bias and filter variants based on the results. By default, strand bias is detected through SOR algorithm.
- Method used to detect strand bias: SOR
- Strand Bias score cutoff: 3
c. Adjacent indel tag
The Adjacent Indel tag is used when a somatic variant was possibly caused by misalignment around a germline or somatic insertion/deletion(indel). By default, CaMutQC will filter any SNV which that was within 10bp of an indel found in the tumor sample. Also, the maximum length of an indel is set as 50bp.
Maximum length of an indel: 50
Minimum interval between an indel and an SNV: 10
d. FILTER tag
Some variant callers add a tag if a variant pass the post-filtration
after calling. In CaMutQC, users can set a standard tag found in the
FILTER column of VCF file to keep variants. PASS is used in
CaMutQC by default.
FILTER tag: PASS
2 Candidate variant selection
a. Database filtration
Some database published germline variants and recurrent artifacts in distinct races. In CaMutQC, based on the parameters we collected 3 4 5, potential germline variants will be removed using annotation from those database(if available) unless the allele frequency of a mutation is lower than the VAF threshold (0.01) or CliVar/OMIM/HGMD flags it as pathogenic.
COSMIC, the
Catalogue Of Somatic Mutations In Cancer, is the world’s largest and
most comprehensive resource for exploring the impact of somatic
mutations in human cancer. They have assembled a list of genes that are
somatically mutated and causally implicated in human cancer 6, which is called the The Cancer Gene Census and is
updated periodically with new genes. In VCF files annotated by VEP, a
Existing_variation column normally indicates a gene is on
this COSMIC list if it has a annotation ID starts with
COSV, COSM or COSN.
Database included: ExAC, Genomesprojects1000, ESP6500, gnomAD
VAF cutoff: 0.01
Keep variants in COSMIC even though they are present in other databases: TRUE
b. Normal depth threshold
To avoid miscalling germline variants and to ensure the quality of variants 4, the filtration for dbsnp/non-dbsnp variants is embedded in CaMutQC with the following cutoffs on normal depth:
- dbsnp Variants: 19
- Non-dbsnp variants: 8
c. Panel of Normals
Panel of Normals or PON is a type of resource used in somatic variant analysis. Basically, if a variant is found in a panel of normals, or is found in more than two normal samples, it is unlikely to be a driven variant during cancer development. PON filtration has been widely used in many researches and projects to discard non-driven variants 3 7 8.
A PON can be generated by users through sequencing a number of normal samples that are as technically similar as possible to the tumor (same exome or genome preparation methods, sequencing technology and so on). Or, a PON can be directly obtained from GATK, which is viewed as one of the most effective filters of false-positive, contamination, and germline variants filter 4.
NCBI build version of this dataset: GRCh38
PON file provided: D:/R-4.5.0/library/CaMutQC/extdata/PON_test.txt
d. Types of variants
Most studies relate to cancer somatic mutations remove certain types
of variants in order to better target candidate variants, among which
exonic and
nonsynonymous are two of the most widely
used categories for filtration 4 9 10.
In CaMutQC, two categories can be chosen during this filtration step.
exonic is the default option, and the
other option is nonsynonymous, it will
leave you non-synonymous variants. More details could be found at Ensembl
Variation.
Variant classifications viewed as exonic:
RNA,Intron,IGR,5\'Flank,3\'Flank,5\'UTR,3\'UTRVariant classifications viewed as nonsynonymous:
3'UTR,5\'UTR,3\'Flank,Targeted_Region,Silent,Intron,RNA,IGR,Splice_Region,5\'Flank,lincRNA,De_novo_Start_InFrame,De_novo_Start_OutOfFrame,Start_Codon_Ins,Start_Codon_SNP,Stop_Codon_Del
Type chose for this filtration: exonic
e. Region selection
In this section, users are able to further select variants related to cancer development by providing a BED file. Variants will be searched only in target regions.
BED file provided: FALSE
3 Filters and flags
| Filter | Flag | Filter | Flag |
|---|---|---|---|
| mutFilterQual | Q | mutFilterPON | P |
| mutFilterSB | S | mutFilterType | T |
| mutFilterAdj | A | mutFilterReg | R |
| mutFilterDB | D | FILTER | F |
| mutFilterNormalDP | N |
4 Target cancer type
In CaMutQC, users are able to filter and select cancer somatic
mutations according to cancer types. mutFilterCan function
integrates ten cancer types so far, with different parameters for each
cancer type, for a more precise and customized filtration.
Parameters in filtration and selection process refer to : Not applied.
5 Study reference
Users are allowed to apply the same filtering strategies that were
used in other studies with CaMutQC, by passing a specific literature
reference as a parameter into the mutFilterRef
function.
Parameters in filtration and selection process refer to : Not applied.
Potential artifacts filtration
Statistics
| Item | Before filtration | After filtration |
|---|---|---|
| # Variants | 87 | 46 |
| # Genes | 72 | 43 |
| Type of variants | Before filtration | After filtration |
|---|---|---|
| SNP | 47 | 24 |
| DNP | 1 | 1 |
| TNP | 0 | 0 |
| ONP | 0 | 0 |
| INS | 6 | 4 |
| DEL | 33 | 17 |
Visualization
1 Flag pie chart
2 Type of variants
3 Distribution of variants in genome
4 VAF Distribution
Candidate variant selection
Statistics
| Item | Before selection | After selection |
|---|---|---|
| # Variants | 46 | 12 |
| # Genes | 43 | 12 |
| Type of variants | Before selection | After selection |
|---|---|---|
| SNP | 24 | 9 |
| DNP | 1 | 1 |
| TNP | 0 | 0 |
| ONP | 0 | 0 |
| INS | 4 | 2 |
| DEL | 17 | 0 |
Visualization
1 Flag pie chart
2 Type of variants
3 Distribution of variants in genome
4 VAF Distribution
TMB
Tumor Mutational Burden (TMB) refers to the number of somatic non-synonymous mutations per megabase pair (Mb) in a specific genomic region. In 2015, tumor non-synonymous mutation burden was first confirmed to be related to PD1/PD-L1 cancer immunotherapy 11. Through the analysis of mutation burden of patients with non-small cell lung cancer, the clinical response and survival rate and other indicators, researchers confirmed that the higher the TMB of cancer patients have, the better the effect of tumor immunotherapy would get. This conclusion was subsequently verified in other cancer types, such as malignant melanoma 12 and small cell lung cancer 13. Therefore, TMB has become one of the predictive biomarkers of immune checkpoint and inhibitor immunotherapy in cancer treatment 14.
There are many calculation methods for TMB, including WGS, WES, regional sequencing using gene panels, and sequencing of circulating tumor DNA in tumor samples or blood 15. Different from scientific research, the conventional method of determining TMB in clinical practice is to target-sequence tumor samples, which is to hybridize and capture the exon and intron regions of a certain number of cancer-related genes, without the need for WES sequencing. Currently, the most widely used panels are FoundationOneCDx (F1CDx) and MSK-IMPACT 9. The former only needs to sequence tumor samples, while the latter requires both the tumor sample and its matched normal sample to be sequenced. Both of them have certification from US Food and Drug Administration (FDA).
In CaMutQC, four methods are supported for TMB calculation, including
FoundationOne, MSK-IMPACT (3 versions of genelist), Pan-cancer panel 16 and WES. By default, TMB is calculated using
MSK-IMPACT method (gene panel version 3, 468 genes). Also, users are
free to apply their own methods by setting parameter assay
as Customized. But it is important to point out
that the bed files used for these assays in CaMutQC (only cover CDS
region of panel genes) are not the real bed region files, and filtration
strategy used may be different, so the result can only be viewed as a
reference.
Method used to calculate TMB: Calculation not asked.
Estimated tumor mutational burden (TMB): Calculation not asked.
Specific Inspection
Variants below are the ones that pass all the filtration functions.
SessionInfo
## R version 4.5.0 (2025-04-11 ucrt)
## Platform: x86_64-w64-mingw32/x64
## Running under: Windows 10 x64 (build 19045)
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## attached base packages:
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